Embryonic Stem cells provide both a unique biological tool for answering developmental questions, but also hold significant clinical promise in the area of regenerative medicine. Murine Embryonic Stem Cells (mESCs), which have existed for almost 25 years, are now being studied extensively for their ability to be maintained in vitro for extended periods of time in a pluripotent state (i.e. able to recreate all the tissues within a mouse). Understanding the mechanisms used to maintain pluripotency may allow for the reprogramming of somatic cells, which opens entirely new treatment options for a variety of diseases. This proposal involves a five-year training plan to foster a career in academic medicine, combining basic science with clinical medicine. This proposal utilizes a combination of stem-cell biology, proteomics, and systems biology to answer questions about mESC pluripotency. It will be performed within the Laboratory of Dr. Stuart Orkin, a leader in the field of stem cell biology who has trained numerous physician scientists. The research project will focus on the transcriptional mechanisms used to maintain mESCs in a pluripotent state. Recent work has shown that a small number of key transcription factors, Nanog, Oct4, and StatS appear to play a key role by regulating a large number of target genes. The mechanisms of transcriptional regulation, which are likely based upon key protein: protein interactions, remains to be elucidated. Initial work has revealed some of the key protein complexes formed in mESCs, although the binary interactions remain to be mapped out. In addition, how these protein: protein interactions occur on a genome-wide scale and affect target gene transcription are currently unknown. For this reason, a systematic yeast two-hybrid approach will be used to first confirm the binary interactions that are used to form these transcriptional complexes. Next, the key complexes formed by STATS will be identified using both new and established proteomics approaches. Lastly, to better understand the protein interactions critical for maintenance of mESCs, multiple yeast two-hybrid screens will be used to generate an "interactome", thereby facilitating a systems biology approach to the problem with significant network analysis. This proposal is ideally performed at the DFCI and CHB in Boston, given the long history of physician-scientist training, but also the unique collaboration with the Center for Cancer Systems Biology. (End of Abstract)